High viscosity xanthan polymer preparations

ABSTRACT

Increasing the molecular length of xanthan polymer makes a higher viscosity xanthan composition. Xanthan with higher specific viscosity characteristics provides more viscosity at equivalent concentration in food, industrial and oilfield applications. Methods for increasing the viscosity of xanthan include inducing particular key genes and increasing copy number of particular key genes.

This application claims the benefit of provisional application U.S. Ser.No. 60/456,245 filed Mar. 21, 2003.

A portion of the disclosure of this patent document contains materialwhich is subject to copyright protection. The copyright owner has noobjection to the facsimile reproduction by anyone of the patent documentor the patent disclosure, as it appears in the Patent and TrademarkOffice patent file or records, but otherwise reserves all copyrightrights whatsoever.

FIELD OF THE INVENTION

The invention relates to the field of microbial products. In particularit relates to microbial products having improved properties for variousindustrial purposes.

BACKGROUND OF THE INVENTION

The chemical structure of xanthan is composed of a linear cellulosic(1→4)-β-D-glucose polymer with trisaccharide side chains composed ofmannose, glucuronic acid and mannose, attached to alternate glucoseresidue in the backbone. (Milas and Rinaudo, Carbohydrate Research, 76,189-196, 1979). Thus xanthan can be described as a branched chainpolymer with a pentasaccharide repeat unit; normal xanthan typically has2000-3000 pentasaccharide repeat units. The xanthan polymer is typicallymodified by acetylation and pyruvylation of the mannose residues.

The fermentation of carbohydrates to produce the biosyntheticwater-soluble polysaccharide xanthan gumBy the action of Xanthomonasbacteria is well known. The earliest work was conducted by the UnitedStates Department of Agriculture and is described in U.S. Pat. No.3,000,790. Xanthomonas hydrophilic colloid (“xanthan”) is an exocellularheteropolysaccharide.

Xanthan is produced by aerobic submerged fermentation of a bacterium ofthe genus Xanthomonas. The fermentation medium typically containscarbohydrate (such as sugar), trace elements and other nutrients. Oncefermentation is complete, the resulting fermentation broth (solution) istypically heat-treated. It is well established that heat treatment ofxanthan fermentation broths and solutions leads to a conformationalchange of native xanthan at or above a transition temperature (T_(M)) toproduce a higher viscosity xanthan. Heat treatment also has thebeneficial effect of destroying viable microorganisms and undesiredenzyme activities in the xanthan. Following heat-treatment, the xanthanis recovered by alcohol precipitation. However, heat treatment ofxanthan fermentation broths also has disadvantages, such as thermaldegradation of the xanthan. Heating xanthan solutions or broths beyondT_(M) or holding them at temperatures above T_(M) for more than a fewseconds leads to thermal degradation of the xanthan. Degradation ofxanthan irreversibly reduces its viscosity. Accordingly, heat treatmentis an important technique with which to control the quality andconsistency of xanthan.

Xanthan quality is primarily determined by two viscosity tests: the LowShear Rate Viscosity (“LSRV”) in tap water solutions and the Sea WaterViscosity (“SWV”) in high salt solutions. Pasteurization of xanthanfermentation broths at temperatures at or above T_(M) has been found toyield xanthan of a higher viscosity as indicated by higher LSRV and SWVvalues.

Xanthan polymer is used in many contexts. Xanthan has a wide variety ofindustrial applications including use in oil well drilling muds, as aviscosity control additive in secondary recovery of petroleum by waterflooding, as a thickener in foods, as a stabilizing agent, and as aemulsifying, suspending and sizing agent (Encyclopedia of PolymerScience and Engineering, 2nd Edition, Editors John Wiley & Sons,901-918, 1989). Xanthan can also be used in cosmetic preparations,pharmaceutical vehicles and similar compositions.

There is a need in the art to produce a xanthan polymer with higherspecific viscosity characteristics in the unpasteurized state. Such ahigher specific viscosity xanthan polymer could provide more viscosityat equivalent xanthan concentrations, for example, for food, industrial,and oilfield applications.

BRIEF SUMMARY OF THE INVENTION

In a first embodiment an unpasteurized xanthan composition is provided.The composition can be provided by a cell which over-expresses gumB andgumC. It has an intrinsic viscosity which is at least 20% greater thanxanthan from a corresponding strain which does not over-express gumB andgumC.

In a second embodiment a xanthan composition is provided. It comprises apopulation of xanthan molecules having a range of molecular lengths. Atleast 1% of the population has a length greater than 3 um as measured byatomic force microscopy.

In a third embodiment of the invention a method is provided forproducing a xanthan polymer preparation having increased viscosityrelative to that produced by a wild-type strain. The amount of geneproduct of gumB and gumC is selectively increased in a Xanthomonascampestris culture. The amount of a gene product of orfX is notselectively increased. Nor is the amount of a product of a gene selectedfrom the group consisting of gumD-gumG selectively increased. A higherviscosity xanthan polymer preparation is thereby produced by theculture.

In a fourth embodiment of the invention a method is provided forproducing a xanthan polymer preparation having increased viscosityrelative to that produced by a wild-type strain. A Xanthomonascampestris strain is cultured in a culture medium under conditions inwhich it produces a xanthan polymer. The strain selectively producesrelative to a wild-type strain more gene product of gumB and gumC butnot of orfX nor of a gene selected from the group consisting ofgumD-gumG.

In a fifth embodiment of the invention an unpasteurized xanthancomposition is provided. The composition is made by a cell whichover-expresses gumB and gumC. The composition has a seawater viscositywhich is at least 10% greater than xanthan from a corresponding strainwhich does not over-express gumB and gumC.

The present invention thus provides the art with xanthan compositionswhich have increased viscosity relative to those similarly produced bycorresponding wild-type strains.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows genetic constructs relative to a genetic map of the gumB-Moperon, also known as the xpsB-M (xanthan polysaccharide synthesis)operon.

FIGS. 2A and 2B show Western blot analyses of gumB and gumC proteinproduct expression, respectively.

FIG. 3 shows an intrinsic viscosity plot for xanthan gum samples, one ofwhich over-expresses gumB and gumC gene products due to the presence ofa plasmid carrying extra copies of the genes.

DETAILED DESCRIPTION OF THE INVENTION

It is a discovery of the present inventors that overexpression of gumBand gumC gene products relative to other genes in their operon, yieldsxanthan products with higher viscosity on a per weight basis. Whileapplicants do not wish to be bound by any particular theory ofoperation, it appears that a shift in the ratio of certain gene productsleads to a shift in the size distribution of xanthan polymer molecules.A significant number of molecules are of higher molecular length thanwhen xanthan is made by a wild-type cell. These longer molecules lead toa higher viscosity of the population or preparation.

It is known in the art that increases in viscosity can be obtained bypasteurizing xanthan preparations. See Talashek et al., U.S. Pat. No.6,391,596. However, the increased viscosity found as the result ofoverexpression of gumB and gumC is observed even in the absence ofpasteurization. Nonetheless subsequent pasteurization of the products ofthe present invention will yield an even more viscous preparation.

Overexpression of both gumB and gumC appear to be required to achievethe increased viscosity. When either gene was tested alone, the increasewas not observed. The overexpression of gumB and gumC can be assessedrelative to other genes of the gumB-M operon. While overexpressionrelative to any of those genes may be sufficient to achieve the effect,overexpression with respect to orfX and gumD may be particularlysignificant. OrfX is a small open reading frame that was previouslypublished as a segment of the genome designated as gumA, immediatelyupstream of gumB. Recently two open reading frames have been discernedin the former gumA region, ihf and orfX Overexpression relative to allof the genes gumD-gumM may be desirable.

Overexpression of the desired gene products may be achieved by any meansknown in the art, including, but not limited to, introducing additionalcopies of the genes encoding the desired gene products to a Xanthomonascampestris cell or other bacterium that makes xanthan, and induction ofthe desired gene products using for example an inducible promoter. Otherbacteria that make xanthan include those that have been geneticallyengineered to contain the xanthan biosynthetic genes. The gumB and gumCgenes can be introduced on one or more vectors, i.e., in combination orindividually.

Inducible promoters which can be used according to the invention includeany that are known in the art, including the lac promoter, the arapromoter, the tet promoter, and the tac promoter. Natural and artificialinducing agents for these promoters are known in the art, and any can beused as is convenient. Additional copies of genes can be introduced onplasmids or viral vectors, for example. Additional copies of the desiredgenes can be maintained extrachromosomally or can be integrated into thegenome.

Recovery of xanthan from a culture broth typically involves one or moreprocessing steps. The xanthan may be heat-treated. The xanthan may beprecipitated with an alchohol, such as isopropyl alcohol, ethyl alcohol,or propyl alcohol. Typically the cells are not specifically removed fromthe culture broth.

Xanthan molecules produced biosynthetically typically have adistribution of sizes. The increased viscosity of the present inventionmay be achieved by increasing the number of molecules having a muchlonger than average length, or by increasing to a greater degree thenumber of molecules having a somewhat longer than average length. Thenumber of molecules which have increased length need not be huge. Atleast 1, 3, 5, 7, 9, or 11% of the molecules with an increased lengthmay be sufficient. The molecules of increased length may be greater than3, 4, 5, 6, 7, 8, or 9 um, as measured by atomic force microscopy. Thepercentage of the mass of the total xanthan population contributed bythe molecules which are longer than 3, 4, 5, 6, 7, 8, or 9 um will begreater than their number proportion in the population. Thus at least 1,3, 5, 10, 15, 20, or 25% of the total mass of the xanthan molecules maybe contributed by molecules having a greater than 3 um length.

Intrinsic viscosity measurements are yet another way to characterize thepreparations of the present invention. Increases seen using this type ofmeasurement may be as great as 5, 10, 15, 20, 25, 30, or 35% over thatproduced by wild-type strains. Proper controls for comparison purposesare those corresponding strains which are most closely related to thestrains being tested. Thus if testing strains that have additionalcopies of gumB and gumC, the best control will have the same geneticcomplement but for the presence of the additional copies of gumB andgumC. If testing cultures that have been induced by an inducer toproduce more gumB and gumC gene product, then the best control will becultures of the same strain that have not been induced. Sea waterviscosity can also be used to characterize preparations of the presentinvention. Increases seen using this type of measurement may be as greatas 5, 10, 15, 20, 25, 30, or 35% over that produced by wild-typestrains.

Xanthan is used as a component in a number of products to improveproperties. The properties may include viscosity, suspension ofparticulates, mouth feel, bulk, to name just a few. Other propertiesinclude water-binding, thickener, emulsion stabilizing, foam enhancing,and sheer-thinning. Such products include foods, such as saladdressings, syrups, juice drinks, and frozen desserts. Such products alsoinclude printing dyes, oil drilling fluids, ceramic glazes, andpharmaceutical compositions. In the latter case, xanthan can be used asa carrier or as a controlled release matrix. Other products wherexanthan can be used include cleaning liquids, paint and ink, wallpaperadhesives, pesticides, toothpastes, and enzyme and cell immobilizers.

While the invention has been described with respect to specific examplesincluding presently preferred modes of carrying out the invention, thoseskilled in the art will appreciate that there are numerous variationsand permutations of the above described systems and techniques that fallwithin the spirit and scope of the invention as set forth in theappended claims.

EXAMPLES Example 1 Strain Construction

To isolate a fragment carrying the complete gum gene region of X.campestris, a genomic library of the wild type X. campestris strain,NRRL B-1459 (1), was constructed with the broad-host-range cosmid vectorpRK311 (2) by cloning of total DNA partially digested with Sau3AI. Thislibrary was mated en masse from E. coli S17-1 (3) to the Gum⁻ X.campestris mutant 2895 (4). One of the cosmids isolated from severalmucoid exconjugants termed pIZD15-261 (5) contains a 16-kb fragmentencompassing the complete gum region. See FIG. 1 for a graphicrepresentation and Table 1 for a listing of the genes of the operon.

TABLE 1 List of genes designations in the chromosomal region encodingxanthan polysaccharide synthesis X. campestris ATCC13951 X. campestris(NRRL B- pv. campestris Chromosomal 1459) ATCC33913 Location* Functioninf himA (XCC2457) 2918744-2918448 integration host factor, alpha chainorfX (XCC2456) 2918464-2918111 transcriptional regulator xpsB gumB(XCC2454) 2917444-2916806 xanthan export xpsC gumC (XCC2453)2916731-2915385 xanthan export xpsD gumD 2915139-2913688 glucosyltransferase (XCC2452) xpsE gumE (XCC2451) 2913602-2912307 xanthanpolymerization xpsF gumF (XCC2450) 2912307-2911216 acetyl transferasexpsG gumG (XCC2449) 2911216-2910149 acetyl transferase xpsH gumH2910078-2908939 mannosyl transferase (XCC2448) xpsI gumI (XCC2447)2908939-2907893 mannosyl transferase xpsJ gumJ (XCC2446) 2907893-2906397xanthan export xpsK gumK (XCC2445) 2906014-2905130 glucuronictransferase xpsL gumL (XCC2444) 2905086-2904295 pyruvyl transferase xpsMgumM 2904284-2903496 glucosyl transferase (XCC2443) orf165 (XCC2442)2903458-2902964 unknown conserved hypothetical *Gene locations areaccording to the genome sequence of X. campestris pv. campestrisATCC33913 (GenBank deposition: AE008922) as described by da Silva, A. C.R., et al., (Nature, Vol. 417, pg. 459-463, 2002)

For the construction of the pBBR5-BC plasmid, a 4026 bp fragment frompIZD15-261 digested with SpeI-BglII was cloned between the XbaI andBamHI sites of pKmob19 (8), giving rise to pGum02-19S (5). A 2855 bpfragment was released from plasmid pGum02-19S by digestion with SphI.This fragment was cloned into pUC 18 (9), which was previously digestedwith SphI, forming pUC18-BCAS.

The final plasmid (pBBR5-BC) was constructed by cloning the HindIII-XbaIfragment, containing the gum promoter and gumB and gumC genes, intoHindIII-XbaI digested pBBR1-MCS5 (10) (GenBank accession no. U25061).

The nucleotide sequence of the resulting pBBR5-BC plasmid is shown inSEQ ID NO: 1. (The predicted amino acid sequences of gumB and gumC areshown in SEQ ID NOs: 2 and 3, respectively. This broad-host-range,medium-copy-number plasmid is 7.6 kb in length and is compatible withIncP, IncQ and IncW group plasmids, as well as with Co1E1- andP15a-based replicons. The presence of an origin of transfer (mobRK2)enables its transference by conjugation into a wide range of bacteriawhen the RK2 transfer functions are provided in trans. It also carriesthe gentamicin resistance gene and it contains the pBluescript II KSmultiple cloning site located within the gene encoding the LacZ apeptide (pBluescript II KS from Stratagene, La Jolla, Calif., USA).

To verify the expression of GumB and GumC proteins from pBBR5-BC, theplasmid was introduced into X. campestris mutant 1231, in which theentire gum (xps) gene cluster was deleted. Both proteins were detectedby Western blot in the mutant strain.

TABLE 2 Bacterial strains and plasmids used or constructed in this work.Source Bacterial strain or plasmid Relevant characteristics (reference)E. coli. DH5α F - endA1 hsdR17 supE44 thi-1 recA1 gyrA relA1 ΔlacU169New England (φ80dlacZΔM15) Biolabs S17-1 E. coli 294RP4-2-Tc::Mu-Km::Tn7 (3) JM109 F′ traD36 proA⁺B⁺ lacl^(q)Δ(lacZ)M15/Δ(lac-proAB) glnV44 e14 New England gyrA96 ompThsdS_(B)(r_(B) ⁻ m_(B) ⁻) gal [dcm] [lon] Biolabs BL2I(DE3) F - ompThsdS_(B)(r_(B) ⁻ m_(B) ⁻) gal [dcm] [lon] (DE3) Novagen X. campestrisNRRL B-1459 Wild type. (1) 2895 Rif^(r) xpsI-261 (11) 1231 Tc::Tn 10ΔxpsI C. P. Kelco XWCM1 Mutant of NRRL B-1459 C. P. Kelco PRM-1 Mutantof NRRL B-1459 C. P. Kelco Plasmids pRK311 oriV(RK2) Tc^(r) oriT(mob⁺)tra⁻ λcos lacZ(α) (2) pIZD15-261 Cosmid based on pRK311 carrying the X.campestris gum (5) region. pK19mob Km^(r), pK19 derivative, mob-site (8)pgum02-19AS pK19mob vector carrying the gum fragment 770-4795^(a) (5)pUC18 Ap^(r), Co1E1, lacZα⁺ (9) pUC18-BCAS pUC18 vector carrying the gumfragment 770-3610^(a) This work pBBR1-MCS5 Gm^(r), pBBR1CM derivative,mob-site, lacZα⁺ (10) pBBR5-BC PBBR1-MCS5 carrying the gum fragment770-3610^(a) This work pQE-Xps#6 pQE30 vector carrying the gum fragment1336-1971^(a) C. P. Kelco pQE30 Ap^(r) Qiagen pREP4 Km^(r) Qiagen pET-CpET22b(+) vector carrying the gum fragment 2135-3319^(a) This workpET22b+ Ap^(r) Novagen pH336 pRK290 carrying gum BamHI fragments1-15052^(a) Synergen pCOS6 pRK293 carrying Sa1I fragments 1-14585a andupstream xps I DNA C P Kelco pFD5 pRK404 carrying partial BamHI gumfragment 318-3464^(a) Ielpi pCHC22 pRK293 carrying Sa1I fragments1-9223a and upstream xps I DNA (4) pBBR-prom pBBR1-MCS5 carrying gumfragment 1000-1276^(a) This work pBBR5-B pBBR1-MCS5 carrying gumfragment 770-1979^(a) This work pBBR-promC pBBR1-MCS5 carrying gumfragment 1979-3459^(a) This work ^(a)Numbers correspond to the positionin the nucleotide sequence of the gum region (GenBank, accession numberU22511)

Bacterial strains, plasmids, and growth conditions. The strains andplasmids used in this study are listed in Table 2. E. coli strains weregrown in Luria-Bertani medium at 37° C. X. campestris strains were grownin TY (5 g of tryptone, 3 g of yeast extract, and 0.7 g of CaCl₂ perliter of H₂O) or in YM medium (12) at 28° C. Antibiotics from Sigma (St.Louis, Mo.) were supplemented as required at the followingconcentrations (in micrograms per milliliter): for X. campestris,gentamicin, 30; and tetracycline, 10; for E. coli, gentamicin, 10;kanamycin, 30; ampicillin, 100; and tetracycline, 10.

DNA biochemistry. Plasmid DNA from E. coli and X. campestris wasprepared by using the QIAprep Spin Miniprep Kit (QIAGEN, Hilden,Germany). DNA restriction, agarose gel electrophoresis and cloningprocedures were carried out in accordance with established protocols(13). All constructs were verified by DNA sequencing. Plasmid DNA wasintroduced into E. coli and X. campestris cells by electroporation asinstructed by Bio-Rad (Richmond, Calif.) (used parameters: E. coli: 200Ω, 25 μF, 2500V and X. campestris: 1000 Ω, 25 μF, 2500V).

Analysis of nucleotide and protein sequences. The nucleotide and aminoacid sequences were analyzed by using the MacVector Sequence AnalysisSoftware (Oxford Molecular Limited, Cambridge, UK).

Example 2 Western Analysis of gumB and gumC Expression

Western Analysis confirmed that gumB and gumC gene products are beingover-expressed in the X. campestris strain with extra copies of gumB andgumC. See FIG. 2.

Example 3 Intrinsic Viscosity Determination

Xanthan samples prepared from X. campestris strains with (XWCM1/pBBR5BC)and without (XWCM1) multiple, plasmid encoded copies of the gumB andgumC genes were compared. Shake flask fermentations, using glucose as acarbon source, were carried out to obtain xanthan from these strains.

Intrinsic viscosity was determined by measuring viscosity on bothpurified and unpurified xanthan samples. An increase in the intrinsicviscosity for xanthan from X. campestris strain with multiple copies ofgumB and gumC was observed. Intrinsic viscosity is proportional to themolecular weight for a given polymer type when measured under identicalsolvent and temperature conditions. Therefore, xanthan from X.campestris strain with multiple copies of gumB and gumC is of highermolecular weight compare to xanthan from control strain.

Methods: Five shake flasks each of the two broths were tested. Thebroths of each type were combined and the total volume measured. Thebroth was then precipitated in isopropyl alcohol. (Note: It wasestimated that the broth contained approximately 3% gum. Measuring thetotal broth volume and multiplying by 3% gave the approximate dry gumweight. This approximation was used to calculate the amount of waterrequired to produce approximately a 0.5% gum solution). The wet fibersof the precipitate were then immediately rehydrated with mixing in 0.01MNaCl to produce approximately a 0.5% gum solution. The fibers were mixedfor three hours with good shear using a 3-blade 2 inch diameterpropeller stirrer, then allowed to stand overnight. The followingprocedure was used to prepare the samples for intrinsic viscositymeasurements.

Filter the ˜0.5% gum solution, prepared above, using a Gelman Science293 mm pressure filtration unit. The solution is first filtered througha 20 μ Magna nylon filter (N22SP29325). The filter is pressurized to ˜60psi, and the solution collected into clean beakers. (Note: the filtersare changed when the flow rate is reduced to ˜5 drips per minute.

Following the first filtration step, the samples are filtered two moretimes using the above filtration unit. First, through a Millipore 8.0 μfilter (SCWP 293 25), then through a Gelman Versapor® 293 mm 1.2 μfilter (66397). The filtered sample is recovered in clean beakersfollowing each filtration step.

After filtration, ˜600 ml of the gum solution is placed intoSpectra/Por® dialysis tubing 28.6 mm diameter Spectrum #S732706 (MWCO12,000 to 14,000). The tubing is cut into lengths of ˜18-20 inches, anda knot tied in one end. The solution is added to the tubing, filling itto within ˜2 inches from the end. Tie a second knot in the tubing suchthat as little air as possible is trapped in the tubing. Continue untilall the gum solution is in dialysis tubing.

Rinse the outside of the tubing containing the gum solution for ˜1minute with de-ionized water, then place the tubing into a container of0.1M NaCl. The salt solution should completely cover the dialysistubing.

Allow the tubing to sit in the 0.01M NaCl solution for 4 days, changingthe NaCl solution daily. After the 4 days, cut open one end of thetubing and carefully transfer the gum solution to a clean beaker.

Solids are run on the filtered dialyzed solution using the followingprocedure:

Using an analytical balance capable of weighing to ±0.0002 g, weigh andrecord the weight of a clean aluminum weighing dish VWR Cat #25433-008.(A)

Using a clean pipet add approximately 10 ml of the gum solution to thealuminum pan and record the exact weight of the combined pan and gumsolution. (B)

Place the pan with the solution into a 105° C. drying oven and allow tostand for 24 hours.

Remove the pan from the oven after 24 hours, cool and reweigh. Recordthe weight of the pan and remaining dried gum. (C)

Subtract the weight of the aluminum pan (A) from the weight of the panplus the gum solution (B). Subtract the weight of the aluminum pan (A)from the weight of the dried gum plus the pan (C). Divide the firstvalue (B-A) into the second (C-A). Multiply this value by 100 to obtainthe % solids.

Note: Solids were run in triplicate for each filtered dialyzed solutionusing the above procedure. The calculated % solids were than averagedfor each sample and the averaged value was used.

Based on the solids determination for each solution, the samples arediluted to 0.25% total gum concentration using 0.01M NaCl.

Intrinsic viscosity measurements were made using the VilasticViscoelasticity Analyzer (Vilastic Scientific, Inc., Austin, Tex.,fitted with the 0.0537 cm radius X 6.137 cm length tube. The instrumentwas calibrated with water prior to making measurements and verifiedafter the measurements were completed. Measurements were conducted usingthe instruments TIMET software protocol, set to a frequency of 2.0 Hz, aconstant strain of 1.0, and an integration time of 10 seconds. Thetemperature was maintained at 23.5° C. The samples were prepared bydilution of the 0.25% gum solution. Each dilution was mixed for 20minutes, and allowed to stand refrigerated overnight before beingmeasured. Six measurements were made for each dilution and averaged.Table 3 below shows the dilutions and the resultant averaged viscositiesfor each prepared sample.

TABLE 3 Viscosity Dilutions Measurements 0.25% X.G. 0.01M NaCl XWCM1XWCM1/ Concentrations (ml) (ml) Control pBBR5-BC Solute 0.01M 0 100 .921.921 NaCl 0.0025% 1 99 1.114 1.165 0.0050% 2 98 1.326 1.486 0.0075% 3 971.537 1.829 0.0100% 4 96 1.762 2.181 0.0150% 6 94 2.302 2.963 0.0200% 892 2.920 3.901

Intrinsic viscosities were determined by plotting the reduced specificviscosity (η_(sp)/c) against the gum concentrationη_(sp)/c=((η_(c)−η_(o))/η_(o)) where η_(c)=viscosity of the gum. Theintercept yields the intrinsic viscosit{tilde over (y)}. See FIG. 3.

The increase in intrinsic viscosity for the XWCM1/pBBR5-BC variant isbelieved due to an increase in molecular weight. Intrinsic viscosity isproportional to the molecular weight for a given polymer type whenmeasured under identical solvent and temperature conditions as done inthis experiment. The relationship between [η] and molecular weight isgiven by the Mark-Houwink equation [η] =kM^(a), where k and a areconstants for a specified polymer type in a specified solvent at aspecified temperature. Because the constant “a” is positive number, anincrease in [η] can only be obtained by an increase in the molecularweight (M) unless the samples have a different molecular conformation inwhich case the Mark-Houwink equation is not obeyed.

Example 4 Procedure—Low Shear Rate Viscosity Measurement

Low shear rate viscosity measurements were performed on purified xanthansamples. The procedure used to measure LSRV is detailed below. Increasedviscosity for xanthan from a strain with multiple copies of gumB andgumC compared to xanthan from a control strain was observed. The datasuggest that over-expression of both gumB and gumC is required forincreased chain length; over-expression of either gumB or gumCindividually is not sufficient to increase chain length.

Material and Equipment:

-   -   1. Standard (synthetic) Tap Water (water containing 1000 ppm        NaCl and 40 ppm Ca⁺⁺ or 147 ppm CaCI₂.2H2O): Prepare by        dissolving in 20 Liters of distilled water contained in a        suitable container, 20 gm of reagent grade NaCl and 2.94 gm of        reagent grade CaCl₂.2H₂O.    -   2. Balance capable of accurately measuring to 0.01 gm.    -   3. Brookfield LV Viscometer, Spindle #1, and spindle Guard.    -   4. Standard laboratory glassware.    -   5. Standard laboratory stirring bench. An RAE stirring motor        (C25U) and stirring shaft ( 5/16″) with 3-bladed propeller may        be substituted.

Procedure:

-   -   1. To 299 ml of synthetic tap water weighed in a 600 ml        Berzelius (tall form) beaker, slowly add 0.75 gm (weighed to the        nearest 0.01 gm) of product,        -   while stirring at 800 rpm.    -   2. After stirring four hours at 800 rpm, remove the solution        from the stirring bench, and allow to stand for 30 minutes.    -   3. Adjust the temperature to room temperature and measure the        viscosity using a Brookfield LV Viscometer with the No. 1        spindle at 3 rpm. Record the viscosity after allowing the        spindle to rotate for 3 minutes.

Example 5 Quantification of Protein Expression

Cell lysates were subjected to Western blot and immunodetection analysisto establish the level of plasmid encoded GumB and GumC. Fourindependent blots were analyzed. Although absolute values for the samesample were not reproducible in each quantification, the relativequantities between samples remained the same in all the measurements.

Preparation of antibodies raised against GumB and GumC. An 1184 bp DNAfragment encoding amino acid residues 53-447 of the GumC protein wasproduced by PCR amplification. The following primers were used: F2135:5′GGAATTCCATATGTTGATGCCCGAGAAGTAC-3′ (SEQ ID NO: 4) and B3319:5′CGGGATCCTCAAAAGATCAGGCCCAACGCGAGG-3 (SEQ ID NO: 5)′. The PCR productwas digested with NdeI and BamHL subcloned into pET22b(+) and theresulting plasmid (pET-C) introduced into the E. coli strain BL21 (DE3).

E. coli BL21(pET-C) grown in L-broth containing 50 μg carbenicillin ml⁻¹to OD₆₀₀ 0.6 was induced with 1 mM IPTG for 3 h. Total cell lysates wereprepared by treating with 1 mg lysozyme ml⁻¹ in lysis buffer (50 mMTris/HCl pH8, 1 mM EDTA pH8, 100 mM NaCl, 1 mM PMSF, 0.1 mg DNaseml^(−1, 0.5)% Triton X-100) at 37° C. for 30 min, followed by sonicationon ice. Cell debris was removed by low speed centrifugation (Eppendof,4000×g, 5 min) and the supernatant was fractionated in a soluble and ina pellet (inclusion bodies) fraction by centrifugation at 14000×g for 10min. Pellet fraction was washed twice with lysis buffer, in a volumeidentical to that of the original cell lysate, once with 2 mg DOC ml⁻¹in lysis buffer followed by three washes with water. After treatment,proteins were separated by SDS-PAGE and the major band containing theoverproduced GumC protein was cut and eluted for immunizing rabbits.

E. coli JM109(pQE-Xps#6, pREP4) grown in L-broth containing 50 μgcarbenicillin, 25 μg kanamycin ml⁻¹ to OD₆₀₀ 0.6 was induced with 1 mMIPTG for 3 h. Total cell lysates were prepared by treating with 1 mglysozyme ml⁻¹ in lysis buffer (50 mM Tris/HCl pH8, 1 mM EDTA pH8, 100 mMNaCl, 1 mM PMSF, 0.1 mg DNase ml⁻¹, 0.5% Triton X-100) at 37° C. for 30min, followed by sonication on ice. Cell debris was removed by low speedcentrifugation (Eppendof, 4000×g, 5 min) and the supernatant wasfractionated in a soluble and in an pellet (inclusion bodies) fractionby centrifugation at 14000×g for 10 min. Pellet fraction was washedtwice with lysis buffer, resuspended in 6 M guanidine hydrochloride in100 mM Phosphate buffer (pH7), 5 mM DTT, 5 mM EDTA and inclusion bodieswere chromatographed on an FPLC Superdex HR200 (Pharmacia Biotech)pre-equilibrated with buffer D (4 M GdnHCl, 50 mM Phosphate buffer(pH7), 150 mM NaCl). Fractions containing GumB were pooled and used toimmunize mice.

Construction of plasmids pFD5, pBBR-promC, and pBBR5-B. A 3141 bpfragment containing gumB and gumC genes was obtained by partialdigestion of pIZD15-261 with BamHI (#318 and #3459) and cloned intoBamHI-digested pRK404 to yield plasmid pFD5. A 1480 bp fragment wasisolated by digestion of pGum02-19 with EcoRI (#1979) and BamHI (#3459)and cloned in pBBR1MCS-5 previously digested with the same enzymes toyield pBBR-promC. Digestion of pGum02-19 with HindIII in the MCS andEcoRI (#1979) produced a 1233 bp fragment, which was cloned inpBBR1MCS-5 to yield plasmid pBBR5-B.

New Zealand white female rabbits were immunized using GumC prepared asdescribed above. A primary injection of 500 μg of the protein withcomplete Freund's adjuvant was given to the rabbits, followed by threeinjections of 250 μg of the protein with incomplete adjuvant onalternate weeks. BALB/c female mice were immunized using GumB preparedas described above. A primary injection of 100 μg of the protein withcomplete Freund's adjuvant was given to the mice, followed by threeinjections of 50 μg of the protein with incomplete adjuvant once a week.Polyclonal antibodies were prepared as described by Harlow & Lane((1999) Using antibodies: a laboratory manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.) and antisera were stored at−70° C. To obtain GumC-specific antibodies, the serum was adsorbed withboth E. coli BL21(pET22b+) and Xc1231 acetone powders (Harlow & Lane,supra).

Protein extracts. Plasmids were introduced into the parental strainPRM-1 by electroporation. The resulting strains were grown in YM mediumat 28° C. and 250 rpm to middle-logarithmic phase. Cells were harvestedby centrifugation and the fresh-weight determined. The pellet was washedtwice with 10 mM Tris/HCl, 10 mM EDTA (pH 8.0) to removeexopolysaccharide and resuspended in the same buffer at a concentrationof 100 mg/ml. After addition of 100 μl Buffer A (10 mM Tris/HCl, 10 mMEDTA (pH 8.0), 1.5% SDS) to 50 μl of each sample, the mixture wasincubated at room temperature for 10 min followed by incubation at 100°C. for 12 min. Cell lysate was centrifuged at 14000×g (Eppendorf 5415 C)for 5 min and the supernatant collected was designated as total proteinextract. Protein concentration of each lysate was determined by themethod of Markwell ((1978) A modification of the Lowry procedure tosimplify protein determination in membrane and lipoprotein samples. AnalBiochem 87(1), 206-10) in the presence of SDS, using BSA as a standard.

SDS-PAGE and inmunodetection. Cell lysates (30 μg per lane) were mixedwith sample buffer (125 mM Tris/HCl, pH6.8; 4% SDS, 20 mM DTT, 0.05%bromophenol blue, 20% glycerol) and boiled for 2 min. Proteins wereseparated by SDS-10% polyacrylamide gel according to the method ofSchagger and von Jagow ((1987) Tricine-sodium dodecylsulfate-polyacrylamide gel electrophoresis for the separation ofproteins in the range from 1 to 100 kDa. Analytical Biochemistry 166(2),368-79). Electroblotting was performed using a semi-dry transfer system(Hoefer Semiphor unit) onto Immobilon-P membranes (PVDF, Millipore). Thetransfer was performed in a buffer containing 10 mM CAPS (pH11), 10%(v/v) methanol for 30 min at 2.5 mA/cm² of gel surface area. Once theelectrotransfer was complete, the blots were stained with 0.5% Ponceau-Sred to assess the quality of the transfer and washed with Milli-Q®-gradewater. The blots were blocked overnight at 4° C. with 5% nonfat milkpowder in TBST (150 mM NaCl, 10 mM Tris/HCl pH8, 0.05% Tween-20) (Harlow& Lane, supra) and then incubated with anti-GumB (1:3000) or anti-GumC(1:5000) antibodies in 3% nonfat milk powder in TBST at room temperaturefor 3 h. Alkaline phosphatase-conjugated goat anti-mouse IgG oranti-rabbit IgG (Sigma) were used for detection, respectively, asdescribed by the manufacturer. The blots were washed three times withTBST and were developed in a solution containing nitrobluetetrazolium-5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP, Promega).Commercial protein markers MW-SDS-70L (Sigma) were used to calibrateSDS-PAGE.

Blot quantification. The intensities of GumB and GumC protein bands weredetermined by scanning the NBT/BCIP developed filters with a UVPDensitometer (Ultra Violet Products) and quantified with GelWorks 1DAnalysis software (NonLinear Dynamics Ltd). Each filter contained areference lane of a PRM-1(pBBR-prom) extract to establish the level ofchromosomally encoded GumB and GumC in the wild type cells. Relativeamounts of GumB and GumC were observed. See FIGS. 2A and 2B.

Example 6 Procedure—Molecular Length or Weight Determination UsingAtomic Force Microscopy

The direct visualization technique called Atomic force Microscopy (AFM)or Scanning Probe Microscope (SPM) was used to image the lengths ofxanthan molecules from X. campestris strains with (XWCM1/pBBR5-BC) andwithout (XWCM1) multiple copies of gumB and gumC. The procedure used toperform AFM is detailed below. We observed that the average molecularcontour length of xanthan molecules produced by a strain with multiplecopies of gumB and gumC was much longer than that of the parentalstrain.

A 0.1 wt % of gum solution was prepared by mixing 0.1 g of gum in 100gram distilled water for ˜3 hours. A 1-ppm stock solution was preparedby diluting 20 μl of the 0.1 wt % solution into a 20 g 0.1M ammoniumacetate solution. 20 μl of the 1 ppm stock solution was sprayed ontofreshly cleaved mica disc(s) (˜1 cm²). These mica sample disc(s) werethen placed in a heated (˜60° C.) vacuum chamber for ˜one hour to removeexcess water. The dried mica disc(s) were then scanned using the TappingMode of the AFM. The molecular contour length of all AFM images wasmeasured with the software provided by Digital Instruments.

Contour lengths of population of xanthan molecules were measured. Theresults of this study are summarized in Table 4. (Molecules in each sizeclass are less than or equal to the length indicated; the number ofmolecules indicated in a size class do not include the molecules countedin a smaller size class.) These results demonstrated that xanthanmolecules from X. campestris strain with multiple copies of gumB andgumC were significantly larger then xanthan molecules from controlstrain. The atomic force microscopy (AFM) or scanning probe microscopy(SPM) was performed with a commercial instrument (Nanoscope IIIa,Digital Instruments, Santa Barbara, Calif.) using a silicon nitridecantilever tip.

TABLE 4 AFM Measurement of Xanthan Molecules Contour Length XWCM1XWCM1/pBBR5-BC Length* Molecules Frequency Distribution Length MoleculesFrequency Distribution (μm) (count) (%) No. Avg. Wt. Avg. (μm) (count)(%) No. Avg. Wt. Avg. 0.5 225 51.5 ≦3 μm = ≦3 μm = 0.5 150 28.4 ≦3 μm =≦3 μm = 1 130 29.7 99.8% 98.7% 1 163 30.9 90.9% 70.9% 1.5 40 9.2 1.5 8215.5 2 25 5.7 2 44 8.3 2.5 13 3.0 2.5 29 5.5 3 3 0.7 3 12 2.3 3.5 00.0 >3 μm = >3 μm = 3.5 12 2.3 >3 μm = >3 μm = 4 0 0.0 0.2% 1.3% 4 132.5 9.1% 29.1% 4.5 0 0.0 4.5 7 1.3 5 1 0.2 5 4 0.8 5.5 0 0.0 5.5 4 0.8 60 0.0 6 0 0.0 6.5 0 0.0 6.5 3 0.6 7 0 0.0 7 2 0.4 7.5 0 0.0 7.5 0 0.0 80 0.0 8 0 0.0 8.5 0 0.0 8.5 1 0.2 9 0 0.0 9 1 0.2 9.5 0 0.0 9.5 1 0.2 100 0.0 10 0 0.0 Total 437 Total 528

Example 7 Evaluation of Seawater Viscosity

Xanthan produced by strain XWCM-1/pBBRS-BC was evaluated for seawaterviscosity (SWV), compared to a commercial xanthan product (Xanvis™).Typical SWV for Xanvis™ xanthan product is in the range of 18 to 22.

Seawater viscosity was determined using the following procedure.Seawater solution was prepared by dissolving 41.95 g of sea salt (ASTMD1141-52, from Lake Products Co., Inc. Maryland Heights, Mo.) in 1 literdeionized water. 300 ml of seawater solution was transferred to a mixingcup that was attached to a Hamilton-Beach 936-2 mixer (Hamilton-BeachDiv., Washington, D.C.). The mixer speed control was set to low and asingle fluted disk attached to the mixing shaft. At the low speedsetting, the mixer shaft rotates at approximately 4,000-6,000 rpm. 0.86g of biogum product was slowly added over 15-30 seconds to the mixingcup and allowed to mix for 5 minutes. The mixer speed control was set tohigh (11,000±1,000 rpm) and the test solution was allowed to mix forapproximately 5 minutes. The mixture was allowed to mix for a total of45 minutes, starting from time of biogum product addition. At the end ofthe 45 minutes mixing time, 2-3 drops of Bara Defoam (NL Baroid/NLindustries, Inc., Houston, Tex.) was added and stirring was continuedfor an additional 30 seconds.

The mixing cup was removed from the mixer and immersed in chilled waterto lower the fluid's temperature to 25±0.5° C. In order to insure ahomogeneous solution, the solution was re-mixed after cooling for 5seconds at 11,000±1,000 rpm. The solution was transferred from themixing cup to 400 ml Pyrex beaker and Fann viscosity (Fann Viscometer,Model 35A) was measured. This was accomplished by mixing at low speed(about 3 rpm). The reading was allowed to stabilize and then the shearstress value was read from dial and recorded as the SW value at 3 rpm.

TABLE 5 Quality of XWCM-1/pBBR5-BC xanthan and Xanvis ™ xanthan SWVSample DR^(a) XWCM-1/pBBR5-BC 29 30 Xanvis xanthan 22 ^(a)dial reading

References

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1. A method of producing a xanthan composition comprising a populationof xanthan molecules having a range of molecular lengths wherein atleast 5% of the population has a length of at least 3 um as measured byatomic force microscopy, comprising: selectively increasing the amountof gene product of wild type gumB and wild type gum C, in a Xanthomonascampestris culture, wherein multiple copies of said wild type gumB andwild type gumC are present but without multiple copes of orfX, gumD,gumE, gumF, and gumG and culturing said Xanthomonas campestris strain.2. A method of producing a xanthan polymer preparation having increasedviscosity relative to that produced by a strain not having amplifiedgenes gumB and gumC, comprising: selectively increasing the amount ofgene product of wild-type gumB and the gene product of wild-type gumC,but not selectively increasing the amount of gene product of orfX andnot selectively increasing the amount of the gene products of gumD,gumE, gumF and gumG in said Xanthomonas campestris strain and culturingsaid Xanthomonas campestris strain, whereby a higher viscosity xanthanpolymer preparation is produced by the culture.
 3. The method of claim 2wherein the step of selectively increasing the amount of gene product ofgumB and gumC is performed by introducing into the Xanthomonascampestris strain one or more additional copies of gumB and gumC.
 4. Themethod of claim 3 wherein the additional copies are on anextrachromosomal genetic element.
 5. The method of claim 4 wherein theextrachromosomal genetic element is a plasmid.
 6. The method of claim 5wherein the plasmid is a broad host range plasmid.
 7. The method ofclaim 2 wherein the additional copies are integrated in the genome ofthe Xanthomonas campestris strain.
 8. The method of claim 2 wherein thestep of selectively increasing the amount of gene product of gumB andgumC is performed by inducing gumB and gumC expression using aninducible promoter and an inducing agent which increases expression fromthe inducible promoter.
 9. The method of claim 2 further comprising thestep of recovering the higher viscosity xanthan polymer from thepreparation.
 10. The method of claim 2 further comprising the step ofprecipitating xanthan polymer from the higher viscosity xanthan polymerpreparation.
 11. The method of claim 1 wherein the Xanthomonascampestris strain comprises one or more plasmids which in aggregatecomprise at least one copy of gumB and gumC.
 12. The method of claim 1further comprising the step of recovering a higher viscosity xanthanpolymer from the culture medium.
 13. The method of claim 1 furthercomprising the step of precipitating xanthan polymer from the culturemedium.